RESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.
Assuntos
Proliferação de Células , Transformação Celular Viral , Modelos Animais de Doenças , Produtos do Gene tax/metabolismo , Infecções por HTLV-I/patologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Linfócitos T/patologia , Animais , Feminino , Perfilação da Expressão Gênica , Produtos do Gene tax/genética , Células HEK293 , Infecções por HTLV-I/metabolismo , Infecções por HTLV-I/virologia , Humanos , Ativação Linfocitária , Masculino , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Domínios PDZ , Ligação Proteica , Linfócitos T/metabolismoRESUMO
UNLABELLED: Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE: Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.
Assuntos
Perfilação da Expressão Gênica , Vírus Linfotrópico T Tipo 3 de Símios/genética , Vírus Linfotrópico T Tipo 3 de Símios/fisiologia , Proteínas Virais/análise , Proteínas Virais/genética , Animais , Linhagem Celular , Núcleo Celular/química , Citosol/química , Humanos , Peso Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/químicaRESUMO
Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteína p300 Associada a E1A/metabolismo , Regulação Neoplásica da Expressão Gênica , Leucemia de Células T/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Telomerase/genética , Proteínas Virais/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Proteína p300 Associada a E1A/genética , Células HeLa , Humanos , Imunoprecipitação , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas dos Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genéticaRESUMO
Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4(+) T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-x(L), and Bcl-2. Indeed, both Bfl-1 and Bcl-x(L) knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-x(L) in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-x(L) represent potential therapeutic targets for ATLL treatment.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Antineoplásicos Fitogênicos/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Compostos de Bifenilo/farmacologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Produtos do Gene tax/genética , Genes jun/genética , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T do Adulto/dietoterapia , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Ligases/genética , Ligases/metabolismo , Masculino , Antígenos de Histocompatibilidade Menor , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Nitrofenóis/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-rel , Proteínas dos Retroviridae , Sulfonamidas/farmacologia , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that Tax(HTLV-1) interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αß T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a "Human Immune System" (HIS) Rag2â»/â»Î³(c)â»/â» mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2â»/â»Î³câ»/â» mice, mature single-positive (SP) CD4⺠and CD8⺠cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2â»/â»Î³(c)â»/â» animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.
Assuntos
Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Timócitos/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Infecções por HTLV-I/patologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia-Linfoma de Células T do Adulto/etiologia , Camundongos , Timócitos/virologia , Quimeras de Transplante/imunologia , Transplante Heterólogo , Carga ViralRESUMO
The study of human pathologies is often limited by the absence of animal models which are robust, cost-effective and reproduce the hallmarks of human infections. While mice have been frequently employed to study human diseases, many of important pathogens display unique human tropism. These last two decades the graft of human progenitor cells or tissues into -immunodeficient mice has allowed the elaboration of so called humanized mice. Humanized mouse technology has made rapid progress, and it is now possible to achieve high levels of human chimerism in various organs and tissues, particularly the immune system and the liver. The review briefly summarizes the different models of humanized mice available for in vivo experiments. With a focus on lymphotropic, monocytotropic and hepatotropic viruses, we here discuss the current status and future prospects of these models for studying the pathogenesis of infectious diseases. Furthermore, they provide a powerful tool for the development of innovative therapies.
Assuntos
Quimera , Doenças Transmissíveis , Modelos Animais de Doenças , Camundongos Mutantes , Animais , Quimera/genética , Quimera/imunologia , Cruzamentos Genéticos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Previsões , Hepatócitos/transplante , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Fígado/embriologia , Transplante de Fígado , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes/genética , Camundongos Mutantes/imunologia , Quimera por Radiação , Especificidade da Espécie , Timo/embriologia , Timo/transplante , Transplante Heterólogo , Viroses/tratamento farmacológico , Viroses/imunologia , Viroses/virologiaRESUMO
Notch and IL-7 are both well-characterized factors involved in T-cell development. In contrast to the mouse model, their precise requirements in the differentiation and/or proliferation of various stages of human thymic development have not been fully explored. Here, we demonstrate that IL-7 alone is sufficient to induce the differentiation of ex vivo purified CD34(+) triple negative (TN) surface (s) CD3(-) CD4(-)CD8(-) (CD3(-)CD4(-)CD8(-)), CD4 immature single positive (ISP) (sCD3(-)CD4(+)CD8(-)) and double positive (DP) (sCD3(-)CD4(+)CD8(+)) human thymic precursors to mature DP expressing sCD3 (sCD3(+)CD4(+)CD8(+)). We show that activation of Notch signaling by its ligands Delta-1 or Delta-4 potentiates IL-7-driven proliferation and survival of CD34(+) TN and to a lesser extent of CD4(+) ISP precursors. This effect of Notch is related to a sustained induction of IL-7 receptor alpha chain expression on thymocytes through a decreased methylation of its gene promoter. Thus, we show here that proliferation and differentiation of T-cell precursors are differentially modulated by IL-7 depending on the presence or absence of external signals. These results may have important implications for the clinical use of this cytokine as a strategy aimed at improving immune restoration.
Assuntos
Diferenciação Celular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-7/imunologia , Proteínas de Membrana/imunologia , Receptores Notch/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD34/imunologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/imunologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Interleucina-7/farmacologia , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/farmacologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacosRESUMO
The palindromic sequence motifs (CANNTG) known as E boxes are considered as binding sites for the basic helix-loop-helix (bHLH) class of DNA-binding proteins. Their presence has been reported in the long terminal repeats (LTR) of the HIV-1 and HTLV-1 proviruses. Their close proximity with the TATA region of both LTRs indicates that the bHLH proteins may act as important regulators of the function of proviral transcription. Indeed, observations on HIV-1 and recent results on HTLV-1 underline that these E boxes may be critically involved in the regulation of the proviral transcription of these human retroviruses. Indeed, of the two E boxes flanking the TATA sequences of the HIV-1 provirus, the 3' E box has been implicated in the transcriptional inhibition of viral gene expression. Such a role might also be played by the unique 5' E box present in the HTLV-1 LTR. In both cases, the expression of tissue-specfic bHLH proteins, like TAL1 might counteract the inhibitory effect exerted by E box proteins, thereby increasing proviral transcription. Finally, a phylogenetic study encompassing several subtypes of these two human retroviruses underlines that these E box motifs have recently appeared in the proviral LTRs and may be considered as potential mediators in the establishment of proviral latency.
Assuntos
Elementos E-Box/fisiologia , Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Provírus/fisiologia , Latência Viral , Evolução Molecular , HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Modelos Biológicos , Filogenia , Provírus/genética , Sequências Repetidas Terminais/genética , Transcrição GênicaRESUMO
The human T-cell leukemia virus type 1 (HTLV-1) Tax transactivator is known to induce or repress various cellular genes, several of them encoding transcription factors. As Tax is known to deregulate various basic bHLH factors, we looked more specifically at its effect on TAL1 (T-cell acute lymphoblastic leukemia 1), also known as SCL (stem cell leukemia). Indeed, TAL1 is deregulated in a high percentage of T-cell acute lymphoblastic leukemia cells, and its oncogenic properties are well-established. Here we show that Tax induces transcription of this proto-oncogene by stimulating the activity of the TAL1 gene promoter 1b, through both the CREB and NF-kappaB pathways. It was also observed that TAL1 upregulates HTLV-1 promoter activity, in either the presence or the absence of Tax. The viral promoter is inhibited in trans by expression of the E2A protein E47, and TAL1 is able to abrogate this inhibition. These data show the existence of a positive feedback loop between Tax and TAL1 expression and support the notion that this proto-oncogene participates in generation of adult T-cell leukemia/lymphoma by increasing the amount of the Tax oncoprotein but also possibly by its own transforming activities.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T do Adulto/virologia , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Linhagem Celular , Retroalimentação Fisiológica , Células HeLa , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Timo/citologiaRESUMO
Articular cartilage (AC) has poor capacities of regeneration and lesions often lead to osteoarthritis. Current AC reconstruction implies autologous chondrocyte implantation which requires tissue sampling and grafting. An alternative approach would be to use scaffolds containing off-the-shelf allogeneic human articular chondrocytes (HACs). To investigate tolerance of allogeneic HACs by the human immune system, we developed a humanized mouse model implanted with allogeneic cartilage constructs generated in vitro. A prerequisite of the study was to identify a scaffold that would not provoke inflammatory reaction in host. Therefore, we first compared the response of hu-mice to two biomaterials used in regenerative medicine, collagen sponge and agarose hydrogel. Four weeks after implantation in hu-mice, acellular collagen sponges, but not acellular agarose hydrogels, showed positive staining for CD3 (T lymphocytes) and CD68 (macrophages), suggesting that collagen scaffold elicits weak inflammatory reaction. These data led us to deepen our evaluation of the biocompatibility of allogeneic tissue-engineered cartilage by using agarose as scaffold. Agarose hydrogels were combined with allogeneic HACs to reconstruct cartilage in vitro. Particular attention was paid to HLA-A2 compatibility between HACs to be grafted and immune human cells of hu-mice: HLA-A2+ or HLA-A2- HACs agarose hydrogels were cultured in the presence of a chondrogenic cocktail and implanted in HLA-A2+ hu-mice. After four weeks implantation and regardless of the HLA-A2 phenotype, chondrocytes were well-differentiated and produced cartilage matrix in agarose. In addition, no sign of T-cell or macrophage infiltration was seen in the cartilaginous constructs and no significant increase in subpopulations of T lymphocytes and monocytes was detected in peripheral blood and spleen. We show for the first time that humanized mouse represents a useful model to investigate human immune responsiveness to tissue-engineered cartilage and our data together indicate that allogeneic cartilage constructs can be suitable for cartilage engineering.
Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Condrogênese , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Osteoartrite/terapia , Transplante HomólogoRESUMO
The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), which are involved in the control of viral gene expression at the transcriptional and post-transcriptional levels. Tax localizes in unique nuclear bodies that contain components of the transcription and splicing complexes. In this work, we studied the relative intracellular localizations of Tax, Rex and p30(II). Run-on transcription assays and immunocytochemistry at light and electron microscopy levels indicated that the Tax nuclear bodies included both de novo transcribed RNA and the RNA polymerase II form that is phosphorylated on its carboxy-terminal domain whereas contacts with chromatin were observed at the periphery of these nuclear bodies. Rex first accumulated in nucleolar foci and then spread across the whole nucleus to display a diffuse and punctuate nucleoplasmic distribution. This distribution of Rex was observed in HTLV-1 transformed lymphocytes and in COS cells expressing the HTLV-1 provirus. Rex colocalized with the cellular export factor CRM-1 in the nucleolar foci as well as in the nucleoplasmic foci that did not overlap with Tax nuclear bodies but were found at the boundaries of the Tax bodies. In addition, we demonstrate that p30(II) interacts with Rex and colocalizes with the Rex/CRM-1 complexes in the nucleoli leading to their clearance from the nucleoplasm. Our results suggest that transcripts originating from Tax-induced activation of gene expression at the boundaries of the Tax bodies are transported out of the nucleus by nucleoplasmic Rex/CRM-1 complexes that are first assembled in nucleolar foci. In addition, p30(II) might exert its negative effect on viral RNA transport by preventing the release of the Rex/CRM-1 complexes from sequestration in nucleolar foci. These data support the idea that the transcriptional and post-transcriptional regulation of HTLV-1 gene expression depends on the concentration of select regulatory complexes at specific area of the nucleus.
Assuntos
Nucléolo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica/genética , Produtos do Gene rex/metabolismo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Proteínas dos Retroviridae/metabolismo , Animais , Células COS , Linhagem Celular , Nucléolo Celular/virologia , Chlorocebus aethiops , Cricetinae , Humanos , Fosforilação , RNA Polimerase II/fisiologia , RNA Viral/metabolismoRESUMO
BACKGROUND: Activation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells display a high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription. RESULTS: Here, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter. CONCLUSION: These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Telomerase/biossíntese , Transcrição Gênica , Regulação para Cima , Proteínas Virais/metabolismo , Imunoprecipitação da Cromatina , DNA/metabolismo , Dimerização , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas dos Retroviridae , Fator de Transcrição Sp1/metabolismo , Telomerase/genéticaRESUMO
BACKGROUND: In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1. RESULTS: We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus. CONCLUSIONS: These observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells.
Assuntos
Transformação Celular Viral , Regulação Viral da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfócitos T/virologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular Transformada , Deleção de Genes , Produtos do Gene rex/genética , Produtos do Gene rex/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/farmacologia , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Humanos , Células Jurkat , Interferência de RNA , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
BACKGROUND: The functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes. RESULTS: We first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax. CONCLUSION: These findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.
Assuntos
Proteínas de Ligação a DNA/genética , Produtos do Gene tax/análise , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Ativação Linfocitária , Linfócitos T/metabolismo , Telomerase/genética , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Transcrição Gênica , Linhagem Celular , Humanos , Complexo Shelterina , Linfócitos T/virologiaRESUMO
The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in the generation of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed.
Assuntos
Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Animais , Humanos , Camundongos , Camundongos SCIDRESUMO
A number of studies implicate reactive oxygen intermediates in the induction of DNA damage and apoptosis. Recent studies suggest that the human T-cell leukemia virus type 1 (HTLV-1) Tax protein induces oxidative stress and apoptotic T-cell death. Activation of the T-cell receptor/CD3 pathway enhances the Tax-mediated oxidative and apoptotic effects. Tax-mediated apoptosis and oxidative stress as well as activation of nuclear factor-kappaB can be potently suppressed by antioxidants. This review focuses on Tax-dependent changes in the intracellular redox status and their role in Tax-mediated DNA damage and apoptosis. The relevance of these observations to HTLV-1 virus-mediated T-cell transformation and leukemogenesis are discussed.
Assuntos
Apoptose/fisiologia , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Linfócitos T/fisiologia , Antioxidantes/metabolismo , Transformação Celular Neoplásica , Humanos , Leucemia-Linfoma de Células T do Adulto/etiologia , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/fisiopatologia , NF-kappa B/metabolismo , Oxirredução , Estresse Oxidativo , Receptores de Antígenos de Linfócitos TRESUMO
Isolated and identified more than 30 years ago, human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma, an aggressive lymphoproliferative disease of activated CD4(+) T cells, and other inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. A variety of animal models have contributed to the fundamental knowledge of HTLV-1 transmission, pathogenesis, and to the design of novel therapies to treat HTLV-1-associated diseases. Small animal models (rabbits, rats, and mice) as well as large animal models (monkeys) have been utilized to significantly advance characterization of the viral proteins and of virus-infected cells in the early steps of infection, as well as in the development of leukemogenic and immunopathogenic processes. Over the past two decades, the creation of new immunocompromised mouse strains that are robustly reconstituted with a functional human immune system (HIS) after being transplanted with human tissues or progenitor cells has revolutionized the in vivo investigation of viral infection and pathogenesis. Recent observations obtained in HTLV-1-infected humanized HIS mice that develop lymphomas provide the opportunity to study the evolution of the proviral clonality in human T cells present in different lymphoid organs. Current progress in the improvement of those humanized models will favor the testing of drugs and the development of targeted therapies against HTLV-1-associated diseases.
RESUMO
The regulatory Tax protein of HTLV-1 (Human T-cell Leukaemia Virus type 1) is critically involved in the initiation of ATL (adult T-cell leukaemia). Indeed, Tax provides infected T-cells with a growth advantage and with the potential to get transformed through the deregulation of cell-cycle progression and the acquisition of genetic alterations. Considering that leukemias are induced by disturbances in hematopoietic cells development, we hypothesize that the expression of Tax in human immature thymocytes is a prerequisite to the emergence of ATL cells. Studies of alph abeta T-cell development in the thymus have shown that beta-selection, an early important checkpoint, is regulated by transcription factors that are decisive in the control of cell proliferation, differentiation and survival. Interestingly, Tax is endowed with the ability to interfere with the activity of these transcription factors. We therefore propose that the HTLV-1 infection of these specific target thymocytes leads to a transcriptional deregulation of early alphabeta T cell development, thus inducing a pre-leukemogenic event that favours the subsequent proliferation of ATL cells.
Assuntos
Transformação Celular Neoplásica , Produtos do Gene tax/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Linfócitos T/citologia , HumanosRESUMO
The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent, and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus, known as beta-selection. E2A transcription factors, which are involved at multiple stages of T-cell development, regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore, we report that in Tax lentivirally transduced human MOLT-4 T cells, which constitutively express the pTalpha gene, the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A, which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally, we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax, by silencing E proteins, down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus.
Assuntos
Regulação para Baixo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Glicoproteínas de Membrana/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Timo/imunologia , Diferenciação Celular/genética , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Luciferases/análise , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Timo/citologiaRESUMO
Tax protein of human T-cell leukaemia virus type 1 (HTLV-1) induces the expression of several cellular genes that are involved in T cell activation and proliferation. In this study, it was observed that Tax upregulated the expression of human interleukin 17 (IL17), a cytokine mainly produced by activated CD4(+) memory T cells. Indeed, IL17 mRNA was highly expressed in HTLV-1-infected T cells as well as in Tax-expressing Jurkat T cells, whereas it was not detectable in HTLV-1-negative T cell lines. The clinical relevance of these observations was further demonstrated by quantitative assessment of IL17 expression in lymphocytes isolated from one HTLV-1-infected patient. To define the transcriptional activation of the IL17 gene by Tax, the 5'-flanking region of this gene was cloned and a reporter gene analysis performed. The presence of a Tax-responsive region spanning 614 bp upstream of the initiation start site was identified, in HeLa as well as in Jurkat cells, stimulated with phorbol myristate acetate and Ca(2+) ionophore. Finally, Tax mutants were used to show that the transcriptional activation of the IL17 promoter by Tax was dependent on the CREB/ATF pathway. As IL17 upregulates the expression of several pro-inflammatory cytokines, these observations provide new insights into the involvement of the Tax protein in the pathophysiology of HTLV-1-associated inflammatory disorders.